A cytogenetic and comparative map of camelid chromosome 36 and the minute in alpacas.

Recent advances in camelid genomics have provided draft sequence assemblies and the first comparative and gene maps for the dromedary (CDR) and the alpaca (LPA). However, no map information is currently available for the smallest camelid autosome-chr36. The chromosome is also of clinical interest because of its involvement in the minute chromosome syndrome (MCS) in infertile alpacas. Here, we developed molecular markers for camelid chr36 by direct sequencing CDR36 and LPA minute and by bioinformatics analysis of alpaca unplaced sequence scaffolds. We constructed a cytogenetic map for chr36 in the alpaca, llama, and dromedary and showed its homology to human chromosome 7 (HSA7) at 49.8-55.5 Mb. The chr36 map comprised seven markers, including two genes-ZPBP and WVC2. Comparative status of HSA7 was further refined by cytogenetic mapping of 16 HSA7 orthologs in camelid chromosomes 7 and 18 and by the analysis of HSA7-conserved synteny blocks across 11 vertebrate species. Finally, mapping chr36 markers in infertile alpacas confirmed that the minute chromosome was a derivative of chr36, but the small size was not a result of a large deletion or a translocation. Instead, cytogenetic mapping of 5.8S, 18S, and 28S rRNA genes (nucleolus organizer region (NOR)) revealed that the size difference between chr36 homologs in infertile alpacas was due to a heterozygous presence of NOR, whereas chr36 in fertile alpacas had no NOR. We theorized that the heterozygous NOR might affect chr36 pairing, recombination, and segregation in meiosis and, thus fertility.

COMPARISON OF CHEMICAL AND SURGICAL VASECTOMY ON TESTICULAR ACTIVITY IN FREE-ROAMING HORSES (EQUUS CABALLUS).

Free-roaming horse (Equus caballus) management is a complex issue incorporating social, economic, emotional, political, and environmental factors. Currently, few proven field techniques exist for managing free-roaming horse population growth, which can reach 20-25% annually. Although there are several strategies available for sterilizing mares when managing free-roaming horse populations, surgical vasectomy is the only method used in the field for stallions. Some managers believe that surgically vasectomizing dominant stallions would have significant effects on reducing horse populations. However, sterilizing only dominant harem stallions results in a relatively modest reduction in population growth as substantial reproduction may occur even when 100% of the dominant harem stallions are sterilized if other males perform as little as 10% of the breeding. The overall goal of the current project was to evaluate the efficacy of a novel nonsurgical method for sterilizing free-roaming horses (chemical vasectomy). In September of 2013, stallions that had been previously surgically vasectomized (SURG, n = 25), previously chemically vasectomized (CHEM, n = 16), or untreated (CONT, n = 32) were captured and surgically castrated in preparation for adoption. When comparing both sterilization methods to CONT, serum testosterone and estrone sulfate concentrations did not differ (P > 0.05), suggesting that these methods for sterilizing free-roaming stallions would not disrupt herd social hierarchy. However, similar to the CONT, all CHEM stallions had sperm present within the vas deferens seminal fluid samples. CHEM stallions had more morphologically abnormal sperm than did CONT stallions but it is not known if this affected the actual fertility. Additional research is needed using alternative sclerosing agents for chemical vasectomy in free-roaming horse populations.

Feeding spent hemp biomass does not adversely affect fertility in rams.

OBJECTIVE: To determine the reproductive effects of feeding spent hemp biomass (SHB) to rams. Several studies suggest cannabidiols negatively affect reproductive characteristics, and the reproductive effects of SHB ingestion have not been investigated in any species. Spent hemp biomass is high in protein and essential fatty acids, indicating a potential use in livestock diets pending studies investigating its safety in animals. METHODS: Polypay rams approximately 6 months old were randomly assigned to 5 feeding trial groups (n = 7/group): either a low or high concentration of SHB in diet for either 4 or 8 weeks plus a control group. Blood samples were collected for hormone assays. At the conclusion of the feeding trial, the testes were removed, and sperm collected directly from the vas deferens were evaluated for motility (total, progressive, and speed), morphology, and concentration. A section from each ram's testes was formalin fixed, paraffin embedded, and subjected to routine immunohistochemistry to determine the expression of fertility-associated proteins deleted in azoospermia-like and Boule, followed by quantitative image analysis. RESULTS: Rams fed either 10% or 20% SHB for 8 weeks had significantly higher progressive sperm motility compared to controls. There were no significant differences between the treatment and control groups in the other parameters studied. Boule immunoexpression was adversely affected, whereas deleted in azoospermia-like immunoexpression was differentially affected by SHB ingestion. CONCLUSION: We conclude that up to 20% of the diet can be fed as SHB to rams for 8 weeks without adversely affecting testicular or sperm function. CLINICAL RELEVANCE: Supplementing young rams with SHB is unlikely to affect fertility.

Canine splenic hemangiosarcoma cells express and activate luteinizing hormone receptors in vitro.

OBJECTIVE: To determine luteinizing hormone receptor (LHR) expression and response to LHR activation in isolated canine splenic hemangiosarcoma cell lines in vitro. SAMPLES: In vitro cultures of commercially available canine splenic hemangiosarcoma cell lines (EFS, GRACE-HSA, and DAL-4). PROCEDURES: The percentage of each cell line expressing LHR was determined by immunocytochemistry. Cells were then treated with increasing doses (7.5 ng/mL, 75 ng/mL) of recombinant canine luteinizing hormone (cLH) for 48 hours and evaluated using a cell proliferation assay. RESULTS: The percentage of cells expressing LHR was 17.2 ± 4.5%, 11.8 ± 3.1%, and 6.9 ± 2.5% in EFS, GRACE-HSA, and DAL-4, respectively. There was significant increase in cell count in the DAL-4 and EFS cell lines following a 48-hour incubation at the highest cLH concentration (P = .028 and P = .019, respectively). There was not a significant increase in cell count in the GRACE-HSA cell line at either cLH concentration. CLINICAL RELEVANCE: Activation of LHR results in cell proliferation in some canine splenic hemangiosarcoma cell lines. These results may explain why spayed and castrated dogs with high circulating LH concentrations may develop hemangiosarcoma more frequently than intact dogs.

The effects of sex and neuter status on trauma survival in dogs: A Veterinary Committee on Trauma registry study.

OBJECTIVE: To determine the effect of sex and neuter status on trauma survival in dogs. DESIGN: Multi-institutional prospective case series, September 2013 to March 2019, retrospectively analyzed. SETTING: Level I and II Veterinary Trauma Centers. ANIMALS: Consecutive sample of 2649 dogs in the American College of Veterinary Emergency and Critical Care Veterinary Committee on Trauma patient registry meeting inclusion criteria. For inclusion, dogs had to have complete data entries, be postpubertal (≥7 months age in females and ≥10 months age in males), and have sustained moderate to severe trauma (animal trauma triage [ATT] score ≥5/18). Dogs that were dead upon arrival, euthanized for financial or unknown reasons alone, or that were presented by a Good Samaritan but subsequently humanely euthanized were excluded. MEASUREMENTS AND MAIN RESULTS: Data collected included age, sex, neuter status (intact, neutered), trauma type (blunt, penetrating, both), outcome (survived to hospital discharge, died, euthanized), and reason for euthanasia (grave prognosis, financial reasons, or both). Of 2649 eligible dogs, 56% survived to hospital discharge (n = 1469). Neutered females had a significantly higher survival rate (58.3% vs 51.3%; P = 0.03) compared to intact females, and neutered males had a significantly higher survival rate (56.6% vs 50.7%; P = 0.04) compared to intact males. There was no significant difference in survival between intact females and intact males (P = 0.87) or between neutered females and neutered males (P = 0.46). Mean cumulative ATT score was higher in intact groups and was found to be a significant predictor of survival (P < 0.01). Based on logistic models, overall odds of survival were 20.7% greater in neutered dogs. CONCLUSIONS: Gonadectomy is associated with lower ATT scores and improved survival after moderate to severe trauma in both female and male dogs.

Restoration of Reproductive Hormone Concentrations in a Male Neutered Dog Improves Health: A Case Study.

This case study reports on the use of hormone therapy to treat a dog with a range of physical and behavioral signs that began after gonadectomy. A male mixed breed dog neutered at 7 months of age presented at 1 year with health issues impacting quality of life. Reduced mobility, limping, rapid weight gain, and fear of unfamiliar people were treated over the next 3 years with trials of pain medication, joint supplements, thyroxine, antidepressant, and significant diet restrictions. Frequent carprofen administration and daily joint supplements reduced limping, but mobility was still poor. Weight stabilized on a strict diet but fear and anxiety responses to strangers continued to worsen. Hormone restoration therapy was initiated when the dog was almost 4 years of age. Weekly subcutaneous administration of testosterone cypionate (0.5 mg/kg) significantly reduced pain and increased muscle mass, thereby improving mobility. However, supraphysiologic concentrations of luteinizing hormone were not reduced with testosterone therapy so a gonadotropin-releasing hormone agonist was implanted. After hormone restoration, appetite was reduced, and anxiety and fear behaviors became manageable. The testosterone and gonadotropin-releasing hormone agonist treatment was easily administered, had no known side effects, and the owners were pleased with the outcome.

Gonad-Sparing Surgical Sterilization in Dogs.

Elective sterilization of pet dogs is a common surgical procedure performed in veterinary practice. The main benefit of sterilization is population control and the reduction in euthanasia of unwanted dogs. The most common methods for sterilizing female and male dogs are ovariohysterectomy (spay; which removes both the ovaries and the uterus) and castration (neutering; which involves removing the testicles), respectively. However, any surgery that removes the gonads changes the animal in both positive and negative ways. There is mounting evidence supporting the long-term health complications associated with surgical sterilization with gonad removal. Gonads are not merely gamete-producing or ancillary sex/reproductive organs but rather they are necessary endocrine glands for normal metabolic, behavioral, musculoskeletal, and anti-neoplastic health. The purpose of this mini review is to describe two gonad sparing surgeries that this author has used to sterilize dogs. These surgeries can be performed on pediatric patients without interfering with pubertal maturation. Dog owners can make the decision when the dog is completely mature whether or not the gonads should be removed.

Possible Relationship between Long-Term Adverse Health Effects of Gonad-Removing Surgical Sterilization and Luteinizing Hormone in Dogs.

Spaying and neutering dogs is commonly used to prevent the birth of unwanted animals and eliminate the risk of reproductive diseases. However, removal of the gonads prevents the feedback of estrogen and testosterone on the pituitary and hypothalamus. As a result, luteinizing hormone (LH) is continuously elevated at supraphysiologic concentrations. Although the main role of LH is for reproductive function (e.g., ovulation), there are LH receptors present in several normal tissues including the thyroid and adrenal glands, gastrointestinal tract, cranial cruciate ligament and round ligament, and lymphocytes. In addition, there are LH receptors present in several neoplastic tissues (e.g., lymphoma, hemangiosarcoma, mastocytoma, transitional cell carcinoma, and osteosarcoma). The role of LH receptors in non-reproductive normal and neoplastic tissues is not known but may stimulate nitric oxide release and induce cell division. The precise etiology of the increased incidence of several non-reproductive long-term health complications following spaying and neutering is not known but may be related to LH receptor activation in these non-reproductive target tissues. How these effects may be mediated is described in this review.

Assessment of luteinizing hormone receptor expression in structural support tissues of canine hip and femorotibial joints.

OBJECTIVE: To determine whether luteinizing hormone receptors (LHRs) are expressed in canine femoral head subchondral bone (FHSB), hip joint round ligament (RL), cranial cruciate ligament (CCL), and femorotibial joint synovium (FJS) specimens. SAMPLE: 1 specimen each of the FHSB, RL, CCL, and FJS obtained from the left hind limbs of 19 fresh canine cadavers. PROCEDURES: 1 section of each FHSB, RL, CCL, and FJS specimen was processed with rabbit polyclonal IgG anti-human LHR antibody, and 1 section was treated with negative control reagents. Percentage immunoexpression of LHRs in FHSB and FJS sections was analyzed by assessment of 100 bone marrow cells or synoviocytes in 3 adjacent hpf (400×). In each RL and CCL section, immunoexpression of LHRs in fibrocytes was semiquantitatively analyzed on the basis of the mean of the product of percentage staining score (from 0 [no staining] to 3 [> 50% of cells stained]) and staining intensity score (from 0 [no staining] to 2 [moderate to strong staining]) for 3 adjacent hpf. RESULTS: All tissues examined had variable LHR expression. Expression of LHRs in FHSB, CCL, or FJS specimens did not differ between sexes or between sexually intact and gonadectomized dogs. However, RL specimens from female dogs had significantly greater LHR expression scores, compared with findings for male dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that LHRs are expressed in structural support tissues of canine hip and femorotibial joints. Further research is required to determine the LHRs' function, mechanism of action, and potential contribution to the pathogenesis of hip dysplasia or CCL rupture in dogs.

Assessment of cytologic evaluation of preputial epithelial cells as a diagnostic test for detection of adrenocortical disease in castrated ferrets.

OBJECTIVE: To determine whether results of cytologic evaluation of preputial epithelial cells correspond to results of a serum endocrine hormone assay and clinical signs associated with adrenocortical disease in castrated ferrets. ANIMALS: 13 clinically normal ferrets and 8 ferrets with signs of adrenocortical disease. PROCEDURES: Blood and preputial lavage samples were collected from each ferret. Serum samples were submitted to the University of Tennessee Veterinary Diagnostic Laboratory for performance of an endocrine hormone assay. Differential epithelial cell counts were performed on preputial lavage samples to determine the percentage of cornified cells. Results of cytologic evaluation were compared with results of the endocrine hormone assay and clinical status of ferrets. RESULTS: The percentage of cornified preputial epithelial cells was not significantly correlated with serum 17B-estradiol or androstenedione concentration but was significantly correlated with serum 17-hydroxyprogesterone concentration (r = 0.60). The percentage of cornified preputial epithelial cells was higher in ferrets with clinical signs of adrenocortical disease (mean +/- SD, 71.3 +/- 16.9%) than in clinically normal ferrets (55.5 +/- 19.0%). CONCLUSIONS AND CLINICAL RELEVANCE: Cornification of preputial epithelial cells was correlated with an increase in serum 17-hydroxyprogesterone concentration as well as clinical signs of adrenocortical disease in castrated ferrets. Additional investigation is needed to elucidate the mechanism of preputial epithelial cell cornification in castrated ferrets.

Platelet aggregation responses in clinically healthy adult llamas.

BACKGROUND: Limited information exists regarding hemostasis in camelids despite the importance of platelet function testing in the accurate identification of platelet disorders. As further importation of llamas to North America is restricted, variability in breeding stock will continue to decrease, potentially leading to an increase in heritable bleeding disorders. OBJECTIVE: The objective of this study was to measure platelet aggregation responses in clinically healthy llamas and provide baseline data to which abnormal platelet function may be compared in the future. METHODS: Blood samples were collected from 39 healthy adult llamas, citrated, and centrifuged to produce platelet-rich plasma (PRP). Within 4 hours of the blood draw, 20 microL of each agonist reagent were added to 180 microL of PRP. Final concentrations of agonists were 2 x 10(-5) M ADP, 0.19 mg collagen/mL PRP, 1 x 10(-4) M epinephrine, and 500 microg arachidonic acid/mL PRP. RESULTS: Llama platelets were most responsive to ADP and collagen, with a maximum percent aggregation (mean+/-SD) of 71.3+/-18.6% and 55.8+/-19% and aggregation rates of 9.5+/-3.9 and 6.7+/-3.7 cm/min, respectively. Llama platelet aggregation in response to epinephrine and arachidonic acid was minimal to absent. CONCLUSIONS: This study is the first of its kind to establish baseline values for platelet aggregation in healthy adult llamas.

Luteinizing hormone receptor expression by nonneoplastic and neoplastic canine lymphocytes.

OBJECTIVE: To investigate luteinizing hormone (LH) receptor expression in canine nonneoplastic and neoplastic lymph nodes, circulating nonneoplastic lymphocytes, and T-cell lymphoma (TCL) cell lines. SAMPLE: Formalin-fixed, paraffin-embedded lymph nodes (5 neoplastic and 3 nonneoplastic) from 6 dogs, circulating lymphocytes from venous blood specimens obtained from 12 healthy dogs, and 3 TCL cell lines derived from 3 dogs with primary lymphoma. PROCEDURES: Lymph node specimens were immunohistochemically stained for determination of LH receptor expression. Circulating nonneoplastic lymphocytes and TCL cell lines were evaluated for LH receptor expression by use of flow cytometry; circulating lymphocytes were also immunophenotyped. The mean percentage of cells positive for LH receptors was determined for each type of specimen. For the healthy dogs, percentages of circulating B and T lymphocytes that expressed LH receptors were assessed on the basis of sex and reproductive status. RESULTS: The mean percentage of LH receptor-positive cells in canine neoplastic and nonneoplastic lymph nodes was 12.4% and 4.1%, respectively. For the healthy dogs, the mean percentage of circulating LH receptor-positive T lymphocytes was significantly higher in gonadectomized dogs (16.6%) than in sexually intact dogs (10.5%); the percentages of circulating LH receptor-positive B lymphocytes did not significantly differ by reproductive status. Among the 3 canine TCL cell lines, LH receptor expression ranged from 10% to 45%. CONCLUSIONS AND CLINICAL RELEVANCE: In this study, LH receptor expression by canine neoplastic and nonneoplastic lymphocytes was detected. Research into the effects of downregulation of LH receptor activation in dogs with lymphoma is warranted.

Effect of different dosages of PG-600 on ovulation and pregnancy rates in ewes during the breeding season.

This study compared the reproductive effects of different dosages of PG-600 (Intervet/Merck Animal Health, Madison, NJ) during the breeding season of ewes. PG-600 is a single-dose injectable product labeled for estrous induction in swine, containing equine chorionic gonadotropin (80 IU/mL) and human chorionic gonadotropin (40 IU/mL). PG-600 is routinely used off-label for out-of-season estrous induction in sheep. However, at the most common dose administered to ewes (5 mL), PG-600 is likely to overstimulate the ovaries, resulting in reduced pregnancy rates. Following estrous synchronization with intravaginal progesterone and cloprostenol, Polypay ewes were treated with 5 mL PG-600 (T1; n = 8), 1.5 mL PG-600 (T2; n = 8), or 5 mL saline (C; n = 8) and then mated to rams. Jugular vein samples were collected prior to the PG-600 injection (0 hr) and at 2, 4, 8, 12, and 24 hr after injection. Serum estradiol-17ß was determined by chemiluminescence and among groups using repeated measures analysis of covariance. Ovulation and pregnancy rates were determined by transrectal ultrasonography and compared by one-way ANOVA and chi-square, respectively. Estradiol-17ß concentrations were greater in T1 compared to T2 and C (P < 0.001). Ovulation rate was greater (P < 0.001) but pregnancy rate was lower (P < 0.001) in the T1 compared to C and T2. These data confirm that a 5 mL dose of PG-600 administered to ewes during the breeding season overstimulates the ovaries, which may then reduce fertilization or embryo survival. Future research will focus on the effects of different dosages of PG-600 on pregnancy rate of ewes during the nonbreeding season.

An Autosomal Translocation 73,XY,t(12;20)(q11;q11) in an Infertile Male Llama (Lama glama) With Teratozoospermia.

Structural chromosome abnormalities, such as translocations and inversions occasionally occur in all livestock species and are typically associated with reproductive and developmental disorders. Curiously, only a few structural chromosome aberrations have been reported in camelids, and most involved sex chromosomes. This can be attributed to a high diploid number (2n = 74) and complex chromosome morphology, which makes unambiguous identification of camelid chromosomes difficult. Additionally, molecular tools for camelid cytogenetics are sparse and have become available only recently. Here we present a case report about an infertile male llama with teratozoospermia and abnormal chromosome number 2n = 73,XY. This llama carries an autosomal translocation of chromosomes 12 and 20, which is the likely cause of defective spermatogenesis and infertility in this individual. Our analysis underlines the power of molecular cytogenetics methods over conventional banding-based chromosome analysis for explicit identification of normal and aberrant chromosomes in camelid karyotypes. This is the first case of a translocation and the first autosomal aberration reported in any camelid species. It is proof of principle that, like in other mammalian species, structural chromosome abnormalities contribute to reproductive disorders in camelids.

Bilateral cystic rete testis in an alpaca (Lama pacos).

A 9-year-old intact male alpaca (Lama pacos) was examined because of marked enlargement of the left scrotum. Ultrasound examination revealed a thin-walled anechoic structure in the area of the left testis. Aspirated fluid contained spermatozoa, many of which had abnormal morphology. Castration was performed and the left testis was markedly enlarged with a clear fluid-filled cyst. The cyst was lined by a single layer of squamous to cuboidal epithelial cells consistent with those originating from rete testis. The right testis was of a comparable size and shape to that of normal alpaca testis, but the rete testis was mildly to moderately dilated. Additional findings included chronic inflammation of the right testis and epididymis and epididymal fibrosis with ductal hyperplasia on the left. The diagnosis was bilateral cystic rete testis, most likely secondary to chronic inflammation.

Alternative methods for feline fertility control: Use of melatonin to suppress reproduction.

PRACTICAL RELEVANCE: Reversible contraceptives are highly desired by purebred cat breeders for managing estrous cycles and by scientists managing assisted reproduction programs. A variety of alternative medicine approaches have been explored as methods to control feline fertility. SCOPE: In the field of veterinary homeopathy, wild carrot seed and papaya have been used for centuries. Both appear to be safe, but their efficacy as feline contraceptives remains anecdotal. In contrast, the use of melatonin in cats has been investigated in a number of studies, findings from which are reviewed in this article. RATIONALE: Cats are seasonally polyestrous (they cycle several times during their breeding season) and are described as long-day breeders because endogenous melatonin negatively regulates estrous cyclicity. Exogenous melatonin administered parenterally also suppresses ovarian activity in cats, and long-term oral or subcutaneous melatonin administration is safe. CHALLENGES: The therapeutic use of melatonin is limited by its short biological half-life (15-20 mins), its poor oral bioavailability and its central effects in reducing wakefulness. Research is required to determine whether higher doses, longer-release formulations, repeated administration or combination implants might overcome these limitations.

Intratesticular and intraepididymal injections to sterilize male cats: From calcium chloride to zinc gluconate and beyond.

AIM AND RATIONALE: The aim of intratesticular and intraepididymal injections is to provide an inexpensive non-surgical method for sterilizing tom cats. Intratesticular and intraepididymal injections have been studied for decades and warrant continued investigation. While both methods result in azoospermia, intratesticular injection of sclerosing agents induces orchitis, resulting in decreased spermatogenesis, whereas intraepididymal injection blocks sperm transport but does not alter spermatogenesis. EVIDENCE BASE: Sclerosing agents that have been used effectively for intratesticular injections in cats include calcium chloride dihydrate and zinc gluconate. For sclerosis by intraepididymal injections, chlorhexidine digluconate has been used successfully in cats. The volume, formulation and concentration of sclerosing agents for intratesticular and intraepididymal injections in cats have not been standardized. CHALLENGES: Neither intratesticular nor intraepididymal injections entirely eliminate gonadal testosterone production, which may be undesirable for pet cats and therefore may restrict the application of this method of sterilization to feral cats with limited human contact. In addition, both methods may require sedation or general anesthesia, leading some to support routine castration over these non-surgical methods. Lastly, even if the technique is successful in inducing permanent sterility, normal fertility may persist in treated males for 1-2 months after treatment because of sperm present within the epididymis and vas deferens.

A novel approach employing ultrasound guidance for percutaneous cardiac muscle injection to retrograde label rat stellate ganglion neurons.

Stellate ganglion (SG) neurons provide the main sympathetic innervation to the heart and help to regulate cardiac function. The purpose of this study was to determine if ultrasound imaging could be employed to retrograde label rat SG neurons innervating the heart without employing thoracotomy. In addition, electrophysiological experiments were performed to characterize the modulation of Ca(2+) channels by neurotransmitters in unlabeled and dye-labeled SG neurons. Fluorescence imaging of actutely isolated cells revealed that dye uptake was successful within five days following injection of dye in the cardiac muscle. Whole-cell voltage-clamp recordings revealed that the majority of the Ca(2+) current was carried by N-type Ca(2+) channels. Finally, fluorescence dye uptake did not appear to affect the modulation of Ca(2+) currents following exposure of SG neurons to norepinephrine, adenosine and neurokinin A. These results demonstrate that ultrasound imaging-guided percutaneous injection can be effectively employed to retrograde label neurons innervating the heart.